Name: a method for remotely silencing neural activity in rodents during discrete phases of learning
Uploaded: 2015-06-22T14:00:00
Description: Watch this Scientific Journal Video about A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning at JoVE.com

Vi takker forfatterne af Robinson et al. 12 for deres bidrag til manuskriptet, hvorfra denne protokol er delvist afledt.

Anvendelse af dyr godkendes af Oberlin College Institutional Animal Care og brug udvalg og er i overensstemmelse med vejledningen for pleje og anvendelse af forsøgsdyr 21. 1. Forberedelse til Viral Infusion Bemærk: Denne protokol bruger BSL-1 forholdsregler. Når der anvendes BSL-2 sikkerhedsforanstaltninger, en engangs kittel, handsker, skoovertræk, øje beskyttelse og et åndedrætsværn (type N95) skal udfyldes. Alle personer, der håndterer BSL-2-...

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Guide for the Care and Use of Laboratory Animals.</a> National Academy Press. , (1996).</li><li>Lowery, R. L., Majewska, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+injection+of+adeno-associated+viral+vectors.">Intracranial injection of adeno-associated viral vectors.</a> J. Vis. Exp. (45), (2010).</li><li>Cavaletti, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+in+the+peripheral+nervous+system+of+systemically+administered+dimethylsulfoxide+in+the+rat:+a+neurophysiological+and+pathological.">Effect in the peripheral nervous system of systemically administered dimethylsulfoxide in the rat: a neurophysiological and pathological.</a> 119 (1-2), 1-2 (2000).</li><li>Parnaudeau, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mediodorsal+thalamus+hypofunction+impairs+flexible+goal-directed+behavior.">Mediodorsal thalamus hypofunction impairs flexible goal-directed behavior.</a> Biol. Psychiatry. 77 (5), 445-453 (2014).</li><li>wiki, D. R. E. A. D. 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Denne protokol beskriver, hvordan man anvender en farmakogenetisk tilgang (DREADD) at undersøge, hvordan en bestemt område af hjernen bidrager til en multi-fase kompleks læring opgave. Med evnen til midlertidigt og eksternt tavshed neurale aktivitet i diskrete områder af hjernen tværs faser af læring, denne kombination af metoder giver en platform til at undersøge en bred vifte af adfærd, herunder mere nuancerede eller maskerede former for læring. I den beskrevne i denne protokol eksempel styre rotter og rotter...

En spændende udfordring inden for adfærdsmæssige neurovidenskab er at bestemme de neurale substrater af komplekse adfærd. En række teknikker såsom permanente læsioner, midlertidig hjerne inaktivering via kanyler implantater og optogenetics har været anvendt til at identificere de bidrag diskrete hjerneområder til underkomponenter komplekse adfærd. Mens disse tilgange informere vores forståelse af regional specificitet under læring, hver teknik er ikke uden begrænsninger. Specifikt permanente læsioner typisk før adfærdsmæssig testning, således deres virkninger er til stede under hele varigheden af ​​paradigme. Kanylering undersøgelser, der involverer præsentationen af en kortsigtet neurale inaktivator (f.eks tetrodotoxin) kan producere betydelig skade på hjernevæv og kan fremkalde stress hos individer lige inden adfærdsmæssige test. Endvidere er inaktivering gennem kanylering begrænset til området af væv, der omgiverspidsen af ​​kanyler. Endelig mens optogenetics tilbyder en række fleksibilitet for den tidsmæssige styring af aktivitet i bestemte områder af hjernen, det er omkostningseffektivt uoverkommelige og teknisk krævende. Disse begrænsninger kan overvindes ved hjælp af en farmakogenetisk tilgang (Designer-Receptorer-Udelukkende-Aktiverede-by-Designer-Drugs, DREADDs) 1,2. Vigtigere er det, mens begrebet farmakogenetik er sofistikeret, udførelsen af ​​teknikken er ligetil. Lig traditionelle stereotaksiske kirurgiske metoder, som involverer infusion af toksin (f.eks NMDA, ibotensyre) i diskrete hjerneområder involverer denne teknik infusion en adeno-associeret virus (AAV), som indeholder et DNA-fragment til et ændret inhiberende G-protein-koblet receptor (hM4Di; designeren receptor) i området af interesse af standard laboratorie gnavere (se figur 1). Den virale vektor indeholder også en fluorescerende reporter (mcitrine). Når indarbejdet itil celler, er designeren receptor (og reporter protein) maksimalt udtrykt ~ 3 uger efter infusion og kan aktiveres selektivt i 2-5 timer ved systemisk administration af den ellers biologisk inert Designerdrug, clozapin-N-oxid (CNO) 1 3. Fordi forsøgslederen er udstyret med præcise, men remote tidsmæssig kontrol over neurale aktivitet i bestemte områder af hjernen, farmakogenetik kombinerer særdeles godt med adfærdsmæssige paradigmer, der er gennemført i flere faser. I dette eksempel, at bidraget fra retrospenialis cortex (RSC) stimulus-stimulus læring i forhold til sin rolle i pavlovsk læring, men denne kombination af metoder er velegnet til et vilkårligt antal spørgsmål, der søger at identificere, hvordan specifikke hjerneområder bidrager til kompleks adfærd. Hertil kommer, mens der ikke er beskrevet i denne protokol, virale og transgene metoder kan bruges til at opnå celletype-specifik DREADD udtryk 2. Som jegs iboende adfærdsmæssige paradigmer, der involverer farmakologiske og / eller andre typer af eksperimentelle manipulationer, nøje overvejelse af eksperimentel design og efterfølgende kvantitativ analyse er påkrævet, når ansætte de DREADD tilgang. Eksperimentatorer nye til DREADD tilgang henvises til en omfattende gennemgang af nuværende DREADD teknologi 2. Hver dag, lære organismer om nye stimuli og begivenheder og deres relationer til hinanden. Selv i et velkendt miljø, såsom hjem, den ene er hurtig til at opdage ændringer i forholdet mellem stimuli, fordi disse ændringer kan være prædiktiv for meningsfulde begivenheder. Sådan stimulus-stimulus (dvs. relationelle) læring involverer konjunktion af multiple stimuli og har traditionelt været forbundet med hippocampus, som er placeret centralt inden i mediale tindingelappen 4. Men hippocampus ikke eksisterer heller handle i isolation; kortikale regioner både inden for og udside af den mediale tindingelappen tilvejebringes en kritisk sensorisk information til hippocampusformationen 5-7. Traditionelle permanente læsion undersøgelser leverer overbevisende dokumentation for inddragelse af en række kortikale regioner (f.eks, de retrospenialis, postrhinal og entorhinal cortex) i hippocampus-afhængig læring, men er begrænset i deres evne til at skelne rollen som en bestemt region under diskrete faser af learning 8-10. Den nuværende protokol tester den hypotese, at RSC er nødvendig for stimulus-stimulus læring ved tavshed RSC i løbet af en enkelt fase af et 3-faset sensorisk konditionering paradigme 11,12. Kort beskrevet rotter modtager infusioner af et AAV, der indeholder designeren receptoren og ~ 3 uger senere indgives den Designerdrug (CNO) 30 min før starten af ​​adfærdsmæssige test. I nærværende protokol, eksperimentelle rotter modtager CNO under den første fase af test (når stimulus-stimulus learning sker), og de modtager køretøjet i løbet af de næste 2 faser i test. For at kontrollere for utilsigtede virkninger af CNO på adfærd, indgyde rotter med designeren receptoren (hM4Di) og injicere med bilen i stedet for CNO. At tage højde for generelle virkninger af viral infusion og receptorekspression, indgyde en kontrol virus, der ikke indeholder designeren receptoren og administrere CNO. Et antal forskellige serotyper af AAV anvendes til at levere genetisk materiale. De nuværende NIH retningslinjer for forskning med rekombinante eller syntetiske molekyler anført, at AAV (alle serotyper) og rekombinante eller syntetiske AAV-konstruktioner, i hvilke transgenet ikke koder enten en potentielt tumorigene genprodukt eller et toksinmolekyle og fremstilles i fravær af en hjælpervirus, kræver BSL-1 forholdsregler (Appendiks B-1. Risk Group 1 (RG1) Agenter) 13. En række anmeldelser vedrørende AAV struktur, bryggers og sikkerhed er til rådighed 14,15. Navnlig, men, På grund af bekymringer vedrørende mulige reproduktive 16,17 og potentielle kræftfremkaldende mekanismer 18-20 i gnavere, nogle institutioner kræver brug af BSL-2 forholdsregler, når du arbejder med AAV. Kontroller den korrekte BSL før brug ved at rådføre sig med tilsyn udvalg på de enkelte institutioner, hvor vil blive udført forskning, Centers for Disease Control og NIH retningslinjer for forskning med rekombinant DNA-molekyler 13 ved brug af virale vektorer for genmanipulation i USA. Personlige værnemidler, investigator uddannelse, vektor indeslutning, dekontaminering, bortskaffelse af dekontamineres materialer, og efter injektion dyr boliger krav specificeres af disse retningslinjer. Desuden høre og følge passende Institutional Animal Care og Brug retningslinjer udvalgsmøder eller tilsvarende institutionelle retningslinier tilsyn udvalgsmøder for at sikre en sikker håndtering, administration og bortskaffelse af AAV.

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	<tbody>
		<tr >
			<td >Male, Long Evans Rats, 55-60 d</td>
			<td >Hilltop Lab Animals Inc</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >rAAV8/hSyn-HA-hM4D(Gi)-IRES-mCitrine</td>
			<td >Virus Vector Core</td>
			<td ></td>
			<td>Caution:&#160; This is a BSL-1 compound</td>
		</tr>
		<tr >
			<td >rAAV8/hSyn-GFP</td>
			<td >Virus Vector Core</td>
			<td ></td>
			<td>Caution:&#160; This is a BSL-1 compound</td>
		</tr>
		<tr >
			<td >Clozapine-N-Oxide</td>
			<td >R&#38;D Systems</td>
			<td >4936-10</td>
			<td>Designer Drug</td>
		</tr>
		<tr >
			<td >Rat Cage lid (Polycarbonate)</td>
			<td >Alternative Design&#160;</td>
			<td >FT 8XL-PC</td>
			<td>Used to cover animal cages 48-72 hours post infusion</td>
		</tr>
		<tr >
			<td >Filer Paper (Replacement)</td>
			<td >Alternative Design&#160;</td>
			<td >FP-R-1018XAD</td>
			<td>Filter paper that goes with cage lids</td>
		</tr>
		<tr >
			<td >Table Top Vise</td>
			<td >JETS</td>
			<td >2201-265</td>
			<td>For holding microscentrifuge tubes containing AAV in the hood</td>
		</tr>
		<tr >
			<td >Medline Biohazard liners&#160;</td>
			<td >Staples</td>
			<td >113444</td>
			<td>Biohazard Trash Bags</td>
		</tr>
		<tr >
			<td >United Solutions 34 gallon rectangular wheeled trashcan with hook and lock handle&#160;</td>
			<td >Amazon.com</td>
			<td ></td>
			<td>Biohazard Trash Can</td>
		</tr>
		<tr >
			<td >Isoflurane, 100 mL</td>
			<td >Patterson Veterinary Supply Inc.&#160;</td>
			<td >07-890-8540</td>
			<td>Anesthetic</td>
		</tr>
		<tr >
			<td >Dual Small Animal Stereotaxic with Digital Display Readout Console</td>
			<td >David Kopf</td>
			<td >Model 942</td>
			<td>Surgical equipment</td>
		</tr>
		<tr >
			<td >Non-rupture Ear Bars, set of 2 (Rat)</td>
			<td >David Kopf</td>
			<td >Model 955</td>
			<td>Surgical equipment</td>
		</tr>
		<tr >
			<td >Anesthesia Mask (Rat)</td>
			<td >David Kopf</td>
			<td >Model 906</td>
			<td>Surgical equipment</td>
		</tr>
		<tr >
			<td >High speed Stereotaxic drill includes table top motor controller, foot pedal, handpiece, stereotaxic handpiece holder</td>
			<td >David Kopf</td>
			<td >Model 1474</td>
			<td>Surgical equipment</td>
		</tr>
		<tr >
			<td >Microdrill burrs, 0.9 mm</td>
			<td >Fine Science Tools Inc</td>
			<td >19007-09</td>
			<td>Surgical supply</td>
		</tr>
		<tr >
			<td >Automated Syringe pump with Micro4 Controller&#160;</td>
			<td >David Kopf</td>
			<td >Model UMP3-1</td>
			<td>Surgical equipment</td>
		</tr>
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			<td >Pro-animal detachable Ceramic Blade Clipper Kit</td>
			<td >Ahdis</td>
			<td >21420</td>
			<td>Surgical supply</td>
		</tr>
		<tr >
			<td >Betadine Skin Cleanser</td>
			<td >Perdue&#160; Products L.P</td>
			<td >67618-149-04</td>
			<td>Surgical supply</td>
		</tr>
		<tr >
			<td >Triple Antiobiotic Ointment</td>
			<td >Medline Supply</td>
			<td >53329-087-01</td>
			<td>Surgical supply</td>
		</tr>
		<tr >
			<td >Puralube Vet Ointment</td>
			<td >Only Veterinary Supply</td>
			<td >17033-211-38</td>
			<td>Surgical supply</td>
		</tr>
		<tr >
			<td >Dino-lite&#160;</td>
			<td >Microscope</td>
			<td >AD7013MTL</td>
			<td>An alternative to the traditional disection scope</td>
		</tr>
		<tr >
			<td >Dino-lite Rigid TableTop Boom Stand</td>
			<td >Microscope</td>
			<td >MS36B</td>
			<td>Surgical equipment</td>
		</tr>
		<tr >
			<td >28 Gauge 10uL Syringe</td>
			<td >Hamilton&#160;</td>
			<td >80308-701SN</td>
			<td>Surgical equipment</td>
		</tr>
		<tr >
			<td >Extra Tall MDF Sound attenuating Cubicle</td>
			<td >Med Associates, Inc</td>
			<td >ENV-018MD</td>
			<td>22&#39;Wx22&#34;Hx16&#34;D</td>
		</tr>
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			<td >Extra Tall Modular Test Chamber</td>
			<td >Med Associates, Inc</td>
			<td >ENV-007</td>
			<td>Behavioral equipment</td>
		</tr>
		<tr >
			<td >Stainless Steel Grid Floor&#160;</td>
			<td >Med Associates, Inc</td>
			<td >ENV-005</td>
			<td>Behavioral equipment</td>
		</tr>
		<tr >
			<td >House Light</td>
			<td >Med Associates, Inc</td>
			<td >ENV-215M</td>
			<td>Used as the house light and stimulus light</td>
		</tr>
		<tr >
			<td >Modular Pellet Dispenser&#160;</td>
			<td >Med Associates, Inc</td>
			<td >ENV-203M-45</td>
			<td>Behavioral equipment</td>
		</tr>
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			<td >Pellet Recetacle, Cup Type</td>
			<td >Med Associates, Inc</td>
			<td >ENV-200R1M</td>
			<td>Behavioral equipment</td>
		</tr>
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			<td >Head Entry Detector for Rat</td>
			<td >Med Associates, Inc</td>
			<td >ENV-254-CB</td>
			<td>Behavioral equipment</td>
		</tr>
		<tr >
			<td >Dustless precision food pellets, 45 mg</td>
			<td >Bio-Serv</td>
			<td >F0165</td>
			<td >Behavioral supply</td>
		</tr>
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			<td >Cage Speaker for Rat Chamber</td>
			<td >Med Associates, Inc</td>
			<td >ENV-224AM</td>
			<td>Behavioral equipment</td>
		</tr>
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			<td >Programmable Audio Generator&#160;</td>
			<td >Med Associates, Inc</td>
			<td >ANL-926</td>
			<td>Behavioral equipment</td>
		</tr>
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			<td >Smart Ctrl 8 Input/16 output Package</td>
			<td >Med Associates, Inc</td>
			<td >DIG-716P2</td>
			<td>Behavioral equipment</td>
		</tr>
		<tr >
			<td >Large Table Top Cabinet and Power Supply</td>
			<td >Med Associates, Inc</td>
			<td >SG-6510D</td>
			<td>Behavioral equipment</td>
		</tr>
		<tr >
			<td >PCI Interface Package</td>
			<td >Med Associates, Inc</td>
			<td >DIG-700P2-R2</td>
			<td>Behavioral equipment</td>
		</tr>
		<tr >
			<td >MED Intel core Computer Pkg with X Pro 19&#34; Monitor</td>
			<td >Med Associates, Inc</td>
			<td >COM-103V</td>
			<td>Behavioral equipment</td>
		</tr>
		<tr >
			<td >Paraformaldehyde (grannular), 1 kg</td>
			<td >Electron Microsopy Sciences</td>
			<td >19210</td>
			<td>Hazard:&#160; carcinogen, weigh in hood</td>
		</tr>
		<tr >
			<td >Rabbit Monoclonal antibody (HA-Tag)</td>
			<td >Cell Signaling Technologies&#160;</td>
			<td >3724S</td>
			<td>Histology reagent</td>
		</tr>
		<tr >
			<td >XP Rabbit monoclonal antibody (GFP)</td>
			<td >Cell Signaling Technologies&#160;</td>
			<td >2956S</td>
			<td>Histology reagent</td>
		</tr>
		<tr >
			<td >Anti-Rabbit IgG</td>
			<td >Cell Signaling Technologies&#160;</td>
			<td >4412S</td>
			<td>Histology supplies</td>
		</tr>
		<tr >
			<td >Superfrost Plus slides&#160;</td>
			<td >VWR international</td>
			<td >483111-703</td>
			<td>Histology supplies</td>
		</tr>
	</tbody>
</table>

a method for remotely silencing neural activity in rodents during discrete phases of learning

Denne protokol beskriver, hvordan man midlertidigt og eksternt tavshed neuronal aktivitet i diskrete hjerneregioner mens dyrene er engageret i indlæring og hukommelse opgaver. Tilgangen kombinerer farmakogenetik (Designer-receptorer-Udelukkende-Aktiveret-by-Designer-Drugs) med en adfærdsmæssig paradigme (sensorisk konditionering), der er designet til at skelne mellem forskellige former for læring. Specifikt viral-medieret levering anvendes til at udtrykke en genetisk modificeret inhiberende G-protein-koblet receptor (Designer Receptor) i et diskret område af hjernen hos gnavere. Tre uger senere, når designer receptor ekspressionsniveauer er høje, er et farmakologisk middel (Designer Drug) administreres systemisk 30 min før en bestemt adfærdsmæssige session. Lægemidlet har affinitet for designeren receptoren og således resulterer i inhibering af neuroner, som udtrykker designeren receptoren, men er ellers biologisk inert. Hjernen regionen forbliver tavshed for 2-5 timer (DEPENding af dosis og administrationsvej). Ved afslutning af det adfærdsmæssige paradigme, er hjernevæv vurderet for korrekt placering og receptorekspression. Denne tilgang er særlig nyttig til bestemmelse af bidraget fra individuelle hjerneregioner til specifikke komponenter i adfærd og kan anvendes på tværs af ethvert antal adfærdsmæssige paradigmer.

Adfærdsmæssige resultater Ved afslutningen af ​​forsøget, bør effektiviteten af ​​områdespecifikke midlertidig inaktivering kvantitativt og kvalitativt vurderet. Det foreliggende eksempel involverer en 3-faset adfærdsmæssige paradigme (sensorisk forkonditionering), hvori CNO blev administreret til at dæmpe neurale aktivitet i RSC Under forkonditioneringen sessioner til at teste den hypotese, at RSC er nødvendigt for dannelsen af foreninger blandt neutrale stimuli 12....

Watch this Scientific Journal Video about A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning at JoVE.com

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while rats are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different components of learning.

En metode til Fjernt Silencing neurale aktivitet i Gnavere Under Diskrete Faser i Learning

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and ...

Research

JoVE Journal

Behavior

Construction of Microdrive Arrays for Chronic Neural Recordings in Awake Behaving Mice

Opførelse af Microdrive Arrays for kroniske Neural Recordings i Awake Behaving Mus

State-of-the-art electrophysiological recordings from the brains of freely behaving animals allow researchers to simultaneously examine local field ...

Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents

Måltid varighed som et mål for Orofacial Nociceptive Svar hos gnavere

A lengthening in meal duration can be used to measure an increase in orofacial mechanical hyperalgesia having similarities to the guarding behavior of ...

Automated Visual Cognitive Tasks for Recording Neural Activity Using a Floor Projection Maze

Automatiserede Visual kognitive opgaver for optagelse neurale aktivitet Brug et gulv Projection Maze

Neuropsychological tasks used in primates to investigate mechanisms of learning and memory are typically visually guided cognitive tasks. We have ...

Measuring Neural and Behavioral Activity During Ongoing Computerized Social Interactions: An Examination of Event-Related Brain Potentials

Måling Neural og adfærdsmæssige aktivitet under Igangværende Edb sociale interaktioner: En undersøgelse af event-relaterede hjernepotentialer

Social exclusion is a complex social phenomenon with powerful negative consequences. Given the impact of social exclusion on mental and emotional health, ...

The Double-H Maze: A Robust Behavioral Test for Learning and Memory in Rodents

Double-H labyrint: En Robust Behavioral Test for Læring og Hukommelse i Gnavere

Spatial cognition research in rodents typically employs the use of maze tasks, whose attributes vary from one maze to the next. These tasks vary by their ...

Quantifying Learning in Young Infants: Tracking Leg Actions During a Discovery-learning Task

Kvantificering Læring i Unge Spædbørn: Tracking Leg Handlinger Under en Discovery-learning Opgave

Task-specific actions emerge from spontaneous movement during infancy. It has been proposed that task-specific actions emerge through a discovery-learning ...

Asymmetric Walkway: A Novel Behavioral Assay for Studying Asymmetric Locomotion

Asymmetrisk Walkway: A Novel Behavioral Assay til at studere Asymmetrisk Locomotion

Behavioral assays are commonly used for the assessment of sensorimotor impairment in the central nervous system (CNS). The most sophisticated methods for ...

Using the Activity-based Anorexia Rodent Model to Study the Neurobiological Basis of Anorexia Nervosa

Brug af aktivitetsbaseret Anoreksi gnaver Model til Undersøgelse Neurobiologisk Grundlag for Anorexia Nervosa

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A ...

A Novel Experimental and Analytical Approach to the Multimodal Neural Decoding of Intent During Social Interaction in Freely-behaving Human Infants

A Novel Eksperimentel og analytisk tilgang til multimodal Neural dekodning hensigtserklæring Under Social Interaktion i frit-opfører Menneskelige Spædbørn

Understanding typical and atypical development remains one of the fundamental questions in developmental human neuroscience. Traditionally, experimental ...